Funktion und Regulation einer neuen Untergruppe von lipidtransportierenden P-Typ ATPasen
Final Report Abstract
Eukaryotic cells are compartmentalized into distinct organelles by lipid bilayers. Assembly and maintenance of the various organellar membranes requires translocation of lipids from one leaflet of the bilayer to the opposing leaflet. Specific membrane proteins, termed lipid flippases, play an essential role in this transport process. P4-ATPases as a subfamily of P-type ATPases represent likely candidates for inward directed ATP-driven lipid transporters. We have shown that in yeast, two members of this subfamily, Dnf1p and Dnf2p, are localized in the plasma membrane and involved in the inward movement of phosphatidylcholine (PC) as well as phosphatidylethanolamine (PE) and phosphatidylserine (PS). Two other subfamily members, Dnf3p and Drs2p, are located in the trans-Golgi network (TGN) and accomplish the enrichment of PS, PE and probably PC in the cytosolic leaflet of post-Golgi secretory vesicles. Collectively, these results indicate that phospholipid asymmetry is created when membrane flows through the Golgi and that P4 ATPases are essential for this process. On the basis of functional complementation experiments and lipid translocation studies in yeast we have shown that functioning of a plant P4-ATPase requires interaction with a novel family of plant membrane-bound proteins. This protein complex is essential for the Golgi machinery involved in secretory processes in peripheral columella cells at the root tip of plants. In parallel, methods for reconstitution of yeast membrane proteins and production of giant unilamellar vesicles have been established. We demonstrated that shape changes of giant unilamellar vesicles can be used as a new tool to study the occurrence and time scale of flippase-mediated transbilayer movement of unlabeled phospholipids. Collectively, this knowledge will facilitate future characterization of P4-ATPases, which, despite being the largest subfamily of P-type ATPases in eukaryotes, remains the least characterized group of primary pumps in all systems.
Publications
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Alder-Baerens, N., Lisman, Q., Luong, L., Pomorski, T. and Holthuis, J.C.M (2006). P4 ATPase-dependent aminophospholipid transport and asymmetry in yeast post-Golgi secretory vesicles. Mol. Biol. Cell 17, 1632-1642.
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Castanys-Muñoz, E., Alder-Baerens, N., Pomorski, T., Gamarro, F. and Castanys, S. (2007) A novel ATP-binding Cassette Transporter from Leishmania is involved in transport of phosphatidylcholine analogs and resistance to alkyl-phospholipids. Mol. Microbiol. 64, 1141-1153.
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Papadopulos, A., Vehring, S., Lopez-Montero, I., Kutschenko, L., Stockl, M., Devaux, P.F., Kozlov, M., Pomorski, T. and Herrmann, A. (2007) Flippase activity detected with unlabelled lipids by shape changes of giant unilamellar vesicles. J. Biol. Chem. 282, 15559-15568.
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Pomorski, T. and Menon, A. K. (2006). Lipid flippases and their biological functions. Cell Mol Life Sci. 63, 2908-2921, invited review.
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Pomorski, T., Holthuis, J.C., Herrmann, A. and van Meer, G. (2004). Tracking down lipid flippases and their biological functions. J. Cell Sci. 117, 805-813.
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Poulsen, L.R., López-Marqués, R.L., McDowell, S.C., Okkeri, J., Licht, D., Pomorski, T., Harper, J.F. and Palmgren, M.G. (2008). A Golgi localized Arabidopsis thaliana P4-ATPase involved in root and shoot development requires a ß subunit to gain functionality. Plant Cell 20, 658-676.
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Vehring, S. and Pomorski, T. (2005). Dynamic processes of phospholipid distribution in the yeast. Invited review, in Cell Biology and Dynamics of Yeast Lipids. Daum, G. (ed). Research Signpost, 85-104.
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Vehring, S., Pakkiri, L., Schröer, A., Alder-Baerens, N., Herrmann, A., Menon, A.K. and Pomorski, T. (2007). Flip-flop of fluorescently labeled phospholipids in proteoliposomes reconstituted with yeast microsomal proteins. Eukaryotic Cell 6, 1625-1634.
